The present invention, in some embodiments thereof, relates to novel glycogen synthase kinase-3 (GSK-3) inhibitors and, more particularly, but not exclusively, to novel substrate-competitive inhibitors of glycogen synthase kinase-3 (GSK-3) and to the use of such inhibitors in the treatment of biological conditions associated with GSK-3 activity.
Protein kinases and phosphorylation cascades are essential for life and play key roles in the regulation of many cellular processes including cell proliferation, cell cycle progression, metabolic homeostasis, transcriptional activation and development. Aberrant regulation of protein phosphorylation underlies many human diseases, and this has prompted the development and design of protein kinase inhibitors. Most of the protein kinase inhibitors developed so far compete with ATP for its binding site. These inhibitors, although often very effective, generally show limited specificity due to the fact that the ATP binding site is highly conserved among protein kinases.
Other sites, such as the substrate's binding site, show more variability in their shape and amino acid compositions and may serve as favorable sites for drug design. Understanding of substrate recognition and specificity is thus essential for development of substrate competitive inhibitors. This knowledge, however, is limited by the scarce amount of structural data regarding substrate binding.
Glycogen synthase kinase-3 (GSK-3) is a constitutively active serine/threonine kinase that modulates diverse cellular functions including metabolism, cell survival and migration, neuronal signaling and embryonic development. Deregulation of GSK-3 activity has been implicated in the pathogenesis of human diseases such as, for example, type-2 diabetes, neurodegenerative disorders and psychiatric disorders. Selective inhibition of GSK-3 is thought to be of therapeutic value in treating these disorders [Bhat et al. (2004). J. Neurochem. 89, 1313-7; Cohen, P. & Goedert, M. (2004). Nat. Rev. Drug Discov. 3, 479-87; Meijer et al. (2004) Trends Pharmacol Sci 25, 471-80; Eldar-Finkelman et al. Biochim Biophys Acta 1804, 598-603; Martinez, A. & Perez, D. I. (2008) J. Alzheimers Dis. 15, 181-91].
Recently, it has been found that GSK-3 is also involved in the pathogenesis of cardiovascular diseases [Cheng et al. 2010 J. Mol Cell Cardiol, in press; Kerkela et al. 2008, J. Clin. Invest. 118:3609-18], of malaria and trypanosomiasis [Droucheau et al. 2004, BBRC, 1700:139-140; Ojo et al. 2008, Antimicrob Agents Chemother, 37107-3717], and in stem cell maintenance or differentiation [Wray et al. 2010 Biochem Soc Trans 1027-32].
In view of the wide implication of GSK-3 in various signaling pathways, development of specific inhibitors for GSK-3 is considered both promising and important regarding various therapeutic interventions as well as basic research.
Some mood stabilizers were found to inhibit GSK-3. However, while the inhibition of GSK-3 both by lithium chloride (LiCl) (WO 97/41854) and by purine inhibitors (WO 98/16528) has been reported, these inhibitors are not specific for GSK-3. In fact, it was shown that these drugs affect multiple signaling pathways, and inhibit other cellular targets, such as inositol monophosphatase (IMpase) and histone deacetylases.
Similarly, an engineered cAMP response element binding protein (CREB), a known substrate of GSK-3, has been described (Fiol et al, 1994), along with other potential GSK-3 peptide inhibitors (Fiol et al, 1990). However, these substrates also only nominally inhibit GSK-3 activity.
Other GSK-3 inhibitors have been reported. Two structurally related small molecules SB-216763 and SB-415286 (GlaxoSmithKline Pharmaceutical) that specifically inhibited GSK-3 were developed and were shown to modulate glycogen metabolism and gene transcription as well as to protect against neuronal death induced by reduction in PI3 kinase activity (Cross et al., 2001; Coghlan et al., 2000). Another study indicated that Induribin, the active ingredient of the traditional Chinese medicine for chronic myelocytic leukemia, is a GSK-3 inhibitor. However, Indirubin also inhibits cyclic-dependent protein kinase-2 (CDK-2) (Damiens et al., 2001). These GSK-3 inhibitors are ATP competitive and were identified by high throughput screening of chemical libraries. It is generally accepted that a major drawback of ATP-competitive inhibitors is their limited specificity (see, for example, Davies et al., 2000).
The present inventors have previously reported of a novel class of substrate competitive inhibitors for GSK-3 [Plotkin et al. (2003) J. Pharmacol. Exp. Ther., 974-980], designed based on the unique substrate-recognition motif of GSK-3 that includes a phosphorylated residue (usually serine) in the context of SXXXS(p) (where S is the target serine, S(p) is phosphorylate serine and X is any amino acid) [see also Woodgett & Cohen (1984) Biochim. Biophys. Acta. 788, 339-47; Fiol et al. (1987) J. Biol. Chem. 262, 14042-8]. Structural studies of GSK-3β identified a likely docking site for the phosphorylated residue; it is a positively charged binding pocket composed of Arg96, Arg180, and Lys205 [Dajani et al. (2001) Cell 105, 721-32; ter Haar et al. (2001) Nature Structural Biology 8, 593-6].
The short phosphorylated peptides patterned after the GSK-3 substrates behaved as substrate competitive inhibitors (Plotkin et al., 2003, supra), with the L803 peptide, KEAPPAPPQS(p)P (SEQ ID NO:4), derived from the substrate heat shock factor-1 (HSF-1) showing the best inhibition activity of those evaluated. An advanced version of L803, the cell permeable peptide L803-mts, was shown to promote beneficial biological activities in conditions associated with diabetes, neuron growth and survival, and mood behavior [Kaidanovich-Beilin & Eldar-Finkelman (2005) J. Pharmacol. Exp. Ther. 316:17-24; Rao et al. (2007) Diabetologia 50, 452-60; Kim et al. (2006) Neuron 52, 981-96; Chen et al. (2004) Faseb J 18, 1162-4; Kaidanovich-Beilin et al. (2004) Biol. Psychiatry. 55:781-4; Shapira et al. (2007) Mol. Cell Neurosci. 34, 571-7].
While further focusing on substrate recognition of GSK-3, three positions in the vicinity of the catalytic site (Phe67 in the P-loop, Gln89 and Asn95) were identified as important for GSK-3 substrates binding [Ilouz et al. (2006) J. Biol. Chem. 281, 30621-30].
Additional background art includes U.S. Pat. Nos. 6,780,625 and 7,378,432; WO 2004/052404 and WO 2005/000192; WO 01/49709; Liberman, Z. & Eldar-Finkelman, H. (2005) J. Biol. Chem. 280, 4422-8; Liberman et al. (2008) Am. J. Physiol. Endocrinol. Metab. 294, E1169-77; Bertrand et al. (2003) J. Mol. Biol. 333, 393-407; Licht-Murava et al., J. Mol. Biol. (2011) 408, 366-378; and Palomo et al. J. Med. Chem. (2012) as published on wwwdotpubsdotacsdotorg as “Just Accepted Manuscript” on Jan. 18, 2012.